Hypoxia, a reduction in tissue oxygen levels below physiologic levels, commonly develops within solid tumors because tumor cell proliferation is greater than the rate of blood vessel formation. Thus, the increase in tumor mass results in aberrant vasculature formation, which compromises the blood supply (Hockel et al., J Natl Cancer Inst 2001 93:266-276). Tumor hypoxia is one stimulus that leads to the increased expression of vascular endothelial growth factor (VEGF) and stimulates angiogenesis, which is essential for meeting the metabolic requirements of tumor growth (Dachs et al., Eur J Cancer 2000 36:1649-1660). In addition, hypoxia contributes to tumor progression to a more malignant phenotype because cells surviving under hypoxic conditions often become resistant to radiotherapy and chemotherapy (Brown, J. M. Cancer Res 1999 59:5863-5870). Thus, factors that regulate the hypoxic events may be good targets for anticancer therapy.
One such target is hypoxia-inducible factor 1 (HIF-1). HIF-1 is a key transcription factor that regulates the blood supply through the expression of vascular endothelial growth factor (VEGF) (Forsythe et al., Mol Cell Biol 1996 16:4604-4613). The biologic activity of HIF-1, a heterodimer composed of HIF-1α and HIF-1β (Wang et al., J Biol Chem 1995 270:1230-1237), depends on the amount of HIF-1α, which is tightly regulated by oxygen tension. Under normoxic conditions, HIF-1α protein is unstable. The instability is mainly regulated by the binding to the von Hippel-Lindau tumor suppressor protein (pVHL) (Maxwell et al., Nature 1999 399:271-275). This binding occurs after the hydroxylation of the two HIF-1α proline residues by HIF-prolyl hyroxylases (Jaakkola et al., Science 2001 292:468-472; Ivan et al., Science 2001 292:464-468; Masson et al., EMBO J. 2001 20:5197-5206). The von Hippel-Lindau protein is one of the components of the multiprotein ubiquitin-E3-ligase complex, which mediates the ubiquitylation of HIF-1α, targeting it for proteasomal proteolysis (Huang et al., Proc Natl Acad Sci USA 1998 95:7987-7992). However, under hypoxic conditions, proline hydroxylation is inhibited, binding between HIF-1 and the von Hippel-Lindau protein is eliminated and HIF-1α becomes stable.
HIF-2α (also known as endothelial PAS protein-1 or MOP2) is another member in HIF family. It was found by homology searches in the gene bank and by cloning experiments. HIF-2α is highly similar to HIF-1α in protein structure, but exhibits restricted tissue-specific expression. HIF-2α is also tightly regulated by oxygen tension and its complex with HIF-1β appears to be directly involved in hypoxic gene regulation, as is HIF-1α. Since HIF-2α is expressed in a number of cancer cell lines and involved in hypoxic gene regulation, HIF-2α is also suggested to be associated with tumor promotion, but may not contribute to the growth of most tumors. In breast cancer cell lines that express both HIF-1α and HIF-2α, HIF-1α rather than HIF-2α appears to predominantly contribute to the transcriptional response to hypoxia. However, HIF-2α may take over the role of HIF-1α in tumors that express only HIF-2α. Indeed, in von Hippel-Lindau (VHL)-defective 786-O renal cell carcinoma cells, the transcriptional response to hypoxia depended on expression levels of HIF-2α. Moreover, the ectopic expression of HIF-2α led to enhanced growth of 786-O tumors grafted in nude mice. Therefore, HIF-2α is also a good target for cancer treatment. See Semenza, G. L., Nature Reviews, Cancer, Vol. 3, (2003), pp. 70-81.
As used herein, the term HIF means the combined effect of or total proteins of HIF-1 plus HIF-2. In addition the term HIF-1 means the combined effect of or total proteins of HIF-1α plus HIF-1β. The term HIF-2 means the combined effect of or total proteins of HIF-2α plus HIF-2β.
While searching for anticancer agents that inhibit HIF-1 activity, we identified a novel pharmacologic action of YC-1 and novel analogs thereof. YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, has been known to inhibit platelet aggregation and vascular contraction by activating soluble guanylyl cyclase, and was originally developed as a potential therapeutic agent for circulation disorders (Teng et al., Eur J Pharmacol 1997 320:161-166; Galle et al., Br J Pharmacol 1999 127:195-203). Recently, we have found two novel biological actions of YC-1 and novel analogs thereof; one is the inhibitory effect on either HIF-1 or HIF-2 activity, and the other is the anti-proliferative effect on cancer cells by arresting the cell cycle and leading to cell apoptosis.
The inhibitory effects of compounds of the invention on the expression of HIF-1α and on the induction of VEGF, aldolase A, and enolase I in cancer cells cultured under hypoxic conditions are also exhibited in vivo, treatment by halting the growth of xenografted tumors originating from human cancers, such as hepatoma, stomach carcinoma, renal carcinoma, cervical carcinoma, neuroblastoma, and prostate carcinoma cells. Tumors from mice treated with the compounds showed fewer blood vessels and reduced expression of HIF-1α protein and HIF-1-regulated genes than tumors from vehicle-treated mice. These results support that the compounds are inhibitors of HIF-1 and HIF-2, and halt tumor growth by blocking tumor angiogenesis and tumor adaptation to hypoxia. The compounds are also useful against tumors that overexpress HIF proteins.
The eukaryotic cell cycle is divided into four stages: G1, S, G2, and M. G1 is the gap phase during which cells prepare for the process of DNA replication. During this phase, cells integrate mitogenic and growth-inhibitory signals and make the decision to proceed, pause, or exit cell cycle. The S Phase is defined as the stage in which DNA synthesis occurs. G2 is the second gap phase during which the cell prepares for the process of division. The M phase is defined as the stage in which the replicated chromosomes are segregated into separate nuclei and other cellular components are divided to make two daughter cells. In addition to G1, S, G2, and M, G0 is defined as the cell stage in which cells exit cell cycle and become quiescent. Cells have evolved signaling pathways to coordinate cell cycle transitions and ensure faithful replication of the genome before cell division. Cell cycle progression is stimulated by protein kinase complexes, each of which consists of a cyclin and a cyclin-dependent kinase (CDK). The CDK's are expressed constitutively through cell cycle, whereas cyclin levels are restricted by transcriptional regulation of the cyclin genes and by ubiquitin-mediated degradation. The CDK activation requires the binding of a cyclin partner in addition to site-specific phosphorylation. To carry on error-free cell cycle, eukaryotic cells have developed control mechanisms that restrain cell cycle transitions in response to stress. These regulatory pathways are termed cell cycle checkpoints, which can be divided into three points, i.e., G1-S, G2, and M phase checkpoint. Cells can arrest transiently at cell cycle checkpoints to allow for the repair of cellular damage. Alternatively, when the cell cycle arrest is due to irreparable damage, checkpoint signaling activates pathways that lead to apoptosis.
In most proliferative disorders, such as benign/malignant tumors, various visceral hyperplasia, vascular wall thickening due to smooth muscle cell proliferation, psoriasis and proliferative retinal diseases, limitless cell proliferation is the most important manifestation. Basically, these disorders are caused by cell cycle dysregulation. Several genes encoding regulatory proteins that govern cell cycle are targets for genetic and epigenetic alterations that underlie the genesis of the proliferative disorders. The best characterized of these genes are D-type cyclins. Amplification of the cyclin D genes occurs in a subset of breast, esophageal, bladder, lung, and squamous cell carcinomas. In addition, cyclin D proteins are over-expressed in some primary tumors and other proliferative disorders. In addition, the catalytic partners of D-type cyclins cdk4 and cdk6 are over-expressed and hyper-activated in some tumors and tumor cell lines. Alterations in other cell cycle regulators have also been implicated in human cancer. Cyclin E has been found to be amplifed, overexpressed, or both in some breast, colon carcinomas, and leukemias. A single instance in which cyclin A was altered in a human hepatoma has been reported. Besides these cell cycle regulators, the genetic alterations of the checkpoint regulators that induce cell cycle arrest are also associated with the genesis of the proliferative disorders.
The p53 gene, whose product plays a key role in checkpoint regulation of cell cycle, is the most frequently mutated gene in human cancer. The stabilization of p53 in response to DNA damage results in enhanced expression of p21, which in turn stops cell cycle at the G1 and G2 phases. This cell cycle arrest makes damaged cells take the time for DNA repair. However, if the DNA damage is irreparable, p53 induces cell death by activating the apoptotic process, which is independent of p21.
Since the disruption of normal cell cycle regulation is the hallmark of cancer, there are numerous opportunities for targeting checkpoint controls to develop new therapeutic strategies for this disease. Such strategies include induction of checkpoint arrest leading to cytostasis and ultimately apoptosis, arrest of proliferating cells in stages of the cell cycle which may sensitize them to treatment with other therapeutic agents such as radiation, and targeting of therapies toward specific regulatory components of the cell cycle. Most anticancer agents intervene at multiple points in the cell cycle. They have diverse mechanisms of action and exhibit specificity in terms of the stage of the cell cycle in which they target i.e., DNA damaging anticancer agents lead to G1/S or G2/M arrest; microtubule targeting agents lead to M arrest; antimetabolites lead to S arrest; and topoisomerase inhibitors lead to S or G2/M arrest. In addition, some potentially successful therapeutic strategies involve the use of agents that target cell cycle regulatory molecules. Chemical inhibitors of cdks, which exhibit specificity for cdk1 and cdk2, can induce both G1 and G2 arrest as well as apoptosis. Therefore, chemicals that specifically cause cell cycle arrest may be useful therapeutic agents for treating cancers and other proliferating disorders irrespective of their target molecules.
The compounds of the invention are also useful for treating non-cancer diseases or conditions which are HIF-mediated or VEGF-mediated. Such diseases or conditions include: atherosclerosis, (Couffinhal et al. Am J Pathol 1997 150:1673-1685); diabetic retinopathy, (Boulton et al. Br J Ophthalmol 1998 82:561-568); cardiac hypertophy, (Kakinuma et al., Circulation 2001 103:2387-23945); vacular remodeling, (Semnza G L, Respir Res 2000 1:159-162); pulmonary hypertension, (Semnza G L, Respir Res 2000 1:159-162); pre-eclampsia, (Caniggia et al., Placenta 2000 21: S25-S30); arthritis, (Anthony et al., Arthritis and Rheumatism 2001 44: 1540-1544); inflammatory disease, (Cramer et al., Cell 2003 112: 645-657); and psoriasis (Bhushan et al., Br. J. Dermatol 1999 141: 1054-1060).
Thus, compounds according to the present invention are useful therapeutic agents, as single agents or when combined with other anticancer therapies, for treating tumors and other proliferative disorders, such as hyper-proliferative skin orders, via inhibition of cell cycle progression.